Uncharacterized Mutations Research Articles

Working with Phage P22.

Transduction experiments in Escherichia coli and Salmonella are usually performed with virulent phage variants. A widely used P1 mutant, called P1 vir, carries one or more uncharacterized mutations that prevent formation of lysogens. In the case of P22, by far the most frequently used variant is named P22 HT105/1 int-201 This phage has a high transducing (HT) frequency due to a mutant nuclease with lower specificity for the pac sequence. As a result, ∼50% of the P22 HT phage heads carry random transducing fragments of chromosomal DNA. The int mutation reduces the formation of stable lysogens. The basic steps in handling the P22 HT105/1 int-201 phage and in performing transduction experiments in Salmonella are described here.

Structure-based prediction of BRAF mutation classes using machine-learning approaches

The BRAF kinase is attracting a lot of attention in oncology as alterations of its amino acid sequence can constitutively activate the MAP kinase signaling pathway, potentially contributing to the malignant transformation of the cell but at the same time rendering it sensitive to targeted therapy. Several pathologic BRAF variants were grouped in three different classes (I, II and III) based on their effects on the protein activity and pathway. Discerning the class of a BRAF mutation permits to adapt the treatment proposed to the patient. However, this information is lacking new and experimentally uncharacterized BRAF mutations detected in a patient biopsy. To overcome this issue, we developed a new in silico tool based on machine learning approaches to predict the potential class of a BRAF missense variant. As class I only involves missense mutations of Val600, we focused on the mutations of classes II and III, which are more diverse and challenging to predict. Using a logistic regression model and features including structural information, we were able to predict the classes of known mutations with an accuracy of 90%. This new and fast predictive tool will help oncologists to tackle potential pathogenic BRAF mutations and to propose the most appropriate treatment for their patients.

A new deep learning technique reveals the exclusive functional contributions of individual cancer mutations

Cancers are caused by genomic alterations that may be inherited, induced by environmental carcinogens, or caused due to random replication errors. Postinduction of carcinogenicity, mutations further propagate and drastically alter the cancer genomes. Although a subset of driver mutations has been identified and characterized to date, most cancer-related somatic mutations are indistinguishable from germline variants or other noncancerous somatic mutations. Thus, such overlap impedes appreciation of many deleterious but previously uncharacterized somatic mutations. The major bottleneck arises due to patient-to-patient variability in mutational profiles, making it difficult to associate specific mutations with a given disease outcome. Here, we describe a newly developed technique Continuous Representation of Codon Switches (CRCS), a deep learning-based method that allows us to generate numerical vector representations of mutations, thereby enabling numerous machine learning-based tasks. We demonstrate three major applications of CRCS; first, we show how CRCS can help detect cancer-related somatic mutations in the absence of matched normal samples, which has applications in cell-free DNA–based assessment of tumor mutation burden. Second, the proposed approach also enables identification and exploration of driver genes; our analyses implicate DMD, RSK4, OFD1, WDR44, and AFF2 as potential cancer drivers. Finally, we used CRCS to score individual mutations in a tumor sample, which was found to be predictive of patient survival in bladder urothelial carcinoma, hepatocellular carcinoma, and lung adenocarcinoma. Taken together, we propose CRCS as a valuable computational tool for analysis of the functional significance of individual cancer mutations.

Smc5/6 Complex Promotes Rad3ATR Checkpoint Signaling at the Perturbed Replication Fork through Sumoylation of the RecQ Helicase Rqh1.

Smc5/6, like cohesin and condensin, is a structural maintenance of chromosomes complex crucial for genome stability. Unlike cohesin and condensin, Smc5/6 carries an essential Nse2 subunit with SUMO E3 ligase activity. While screening for new DNA replication checkpoint mutants in fission yeast, we have identified two previously uncharacterized mutants in Smc5/6. Characterization of the mutants and a series of previously reported Smc5/6 mutants uncovered that sumoylation of the RecQ helicase Rqh1 by Nse2 facilitates the checkpoint signaling at the replication fork. We found that mutations that eliminate the sumoylation sites or the helicase activity of Rqh1 compromised the checkpoint signaling similar to a nse2 mutant lacking the ligase activity. Surprisingly, introducing a sumoylation site mutation to a helicase-inactive rqh1 mutant promoted cell survival under stress. These findings, together with other genetic data, support a mechanism that sumoylation of Rqh1 by Smc5/6-Nse2 recruits Rqh1 or modulates its helicase activity at the fork to facilitate the checkpoint signaling. Since the Smc5/6 complex, Rqh1, and the replication checkpoint are conserved in eukaryotes, a similar checkpoint mechanism may be operating in human cells.

Distinct genomic landscape of Chinese pediatric acute myeloid leukemia impacts clinical risk classification

Studies have revealed key genomic aberrations in pediatric acute myeloid leukemia (AML) based on Western populations. It is unknown to what extent the current genomic findings represent populations with different ethnic backgrounds. Here we present the genomic landscape of driver alterations of Chinese pediatric AML and discover previously undescribed genomic aberrations, including the XPO1-TNRC18 fusion. Comprehensively comparing between the Chinese and Western AML cohorts reveal a substantially distinct genomic alteration profile. For example, Chinese AML patients more commonly exhibit mutations in KIT and CSF3R, and less frequently mutated of genes in the RAS signaling pathway. These differences in mutation frequencies lead to the detection of previously uncharacterized co-occurring mutation pairs. Importantly, the distinct driver profile is clinical relevant. We propose a refined prognosis risk classification model which better reflected the adverse event risk for Chinese AML patients. These results emphasize the importance of genetic background in precision medicine.

Neutralization Sensitivity of HIV-1 CRF07_BC From an Untreated Patient With a Focus on Evolution Over Time.

The diversity of HIV-1 envelope (Env) glycoproteins affects the potency and breadth of broadly neutralizing antibodies (bNAbs), a promising alternative to antiretroviral drugs for the prevention and treatment of HIV-1 infection. To facilitate immunogen design and development of therapeutic neutralizing antibodies, we characterized viral evolution and monitored the changes in neutralizing activity/sensitivity of a long-term non-progressor patient with HIV-1 CRF07_BC infection. Fifty-nine full-length Env gene fragments were derived from four plasma samples sequentially harvested from the patient between 2016 and 2020. Sequencing of patient-derived Env genes revealed that potential N-linked glycosylation sites (PNGS) in V1 and V5 significantly increased over time. Further, 24 functional Env-pseudotyped viruses were generated based on Env gene sequences. While all 24 Env-pseudotyped viruses remained sensitive to concurrent and subsequent autologous plasma, as well as bNAbs, including 10E8, VRC01, and 12A21, Env-pseudotyped viruses corresponding to later sampling time were increasingly more resistant to autologous plasma and bNAbs. All 24 Env-pseudotyped viruses were resistant to bNAbs 2G12, PGT121, and PGT135. The neutralization breadth of plasma from all four sequential samples was 100% against the global HIV-1 reference panel. Immune escape mutants resulted in increased resistance to bNAb targeting of different epitopes. Our study identified known mutations F277W in gp41 and previously uncharacterized mutation S465T in V5 which may be associated with increased viral resistance to bNAbs.

Familial Essential Thrombocythemia With Novel MPL L502G and G208K Mutations

Familial essential thrombocythemia is characterized by the inheritance of germline mutations to progeny, thereby increasing the risk for the development of essential thrombocythemia. Here, we present two cases of young women who developed thromboembolic phenomena, one of whom with an ischemic event despite adequate anticoagulation. Through extended mutational testing, both were characterized as having novel mutations in the myeloproliferative leukemia virus (MPL) gene, and both individuals have fathers being treated for essential thrombocythemia. This case provides insight that in familial essential thrombocythemia, there remain uncharacterized mutations in this inherited conditional landscape.

Base editing sensor libraries for high-throughput engineering and functional analysis of cancer-associated single nucleotide variants.

Base editing (BE) can be applied to characterize single nucleotide variants (SNVs) of unknown function, yet defining effective combinations of single guide RNAs (sgRNAs) and base editors remains challenging. Here, we describe modular BE-activity ‘sensors’ that link sgRNAs and cognate target sites in cis and use them to systematically measure the editing efficiency and precision of thousands of sgRNAs paired with functionally distinct base editors. By quantifying sensor editing across >200,000 editor–sgRNA combinations, we provide a comprehensive resource of sgRNAs for introducing and interrogating cancer-associated SNVs in multiple model systems. We demonstrate that sensor-validated tools streamline production of in vivo cancer models, and that integrating sensor modules in pooled sgRNA libraries can aid interpretation of high-throughput BE screens. Using this approach, we identify several previously uncharacterized mutant TP53 alleles as drivers of cancer cell proliferation and in vivo tumor development. We anticipate that the framework described here will facilitate the functional interrogation of cancer variants in cell and animal models.

Two uncharacterized point mutations in the cardiac Kv7.1 channel influence its conductance and lead to LQTS in the patient

Cation channels Kv7.1 encoded by KCNQ1 genes are vital for the formation of cardiac action potential. Malfunctions in the Kv7.1 channel lead to inherited arrhythmias. Loss-of-function mutations slow membrane repolarization whichappears as QTc interval prolongation on standard ECG (LQTS). The biophysical effect of novel and non-characterized genetic variants requires careful functional characterization for correct pathogenicity assessment.

Sulfonylurea-Insensitive Permanent Neonatal Diabetes Caused by a Severe Gain-of-Function Tyr330His Substitution in Kir6.2

Background/Aims: Mutations in KCNJ11, the gene encoding the Kir6.2 subunit of pancreatic and neuronal K_ATP channels, are associated with a spectrum of neonatal diabetes diseases. Methods: Variant screening was used to identify the cause of neonatal diabetes, and continuous glucose monitoring was used to assess effectiveness of sulfonylurea treatment. Electrophysiological analysis of variant K_ATP channel function was used to determine molecular basis. Results: We identified a previously uncharacterized KCNJ11 mutation, c.988T>C [p.Tyr330His], in an Italian child diagnosed with sulfonylurea-resistant permanent neonatal diabetes and developmental delay (intermediate DEND). Functional analysis of recombinant K_ATP channels reveals that this mutation causes a drastic gain-of-function, due to a reduction in ATP inhibition. Further, we demonstrate that the Tyr330His substitution causes a significant decrease in sensitivity to the sulfonylurea, glibenclamide. Conclusions: In this subject, the KCNJ11 (c.988T>C) mutation provoked neonatal diabetes, with mild developmental delay, which was insensitive to correction by sulfonylurea therapy. This is explained by the molecular loss of sulfonylurea sensitivity conferred by the Tyr330His substitution and highlights the need for molecular analysis of such mutations.

In silico analysis of a SLC6A4 G100V mutation in lung cancers.

SLC6A4 is a serotonin re-uptake transporter which has been a target for anti-depressant therapies but recently some mutations have been described in cancer cells. Here, we characterize mutations in SLC6A4 that appear in cancer cells. We employed several validated computational and artificial intelligence algorithms to characterize the mutations. We identified a previously uncharacterized G100V mutation in lung cancers. In sillico structural analysis reveals that this mutation may affect SLC6A4 ligand binding and subsequently its function. We also identified several other mutations that may affect the structure of the protein. This preliminary analysis highlights the role of SLC6A4 in human cancers.

Target-site resistance to trifluralin is more prevalent in annual ryegrass populations from Western Australia.

Trifluralin is widely used in Australia as one of the important pre-emergence herbicides to control annual ryegrass (Lolium rigidum Gaud.) populations. Trifluralin resistance evolution and mechanisms have been identified in some ryegrass populations. In this study, 21 putative resistant field survey populations from Western Australian were screened with trifluralin, and 90% (19 of 21) contained individuals surviving 480 g ha-1 trifluralin treatment. Twelve populations contained individuals possessing the known α-tubulin resistance mutations at Val-202, Thr-239 and Arg-243 in TUA4 (alpha-tubulin 4 n), plus multiple potential resistance mutations in TUA4 pending genetic confirmation. Three populations had only individuals carrying newly identified (but uncharacterized) mutations in TUA3/TUA4. Radioactive work found that six populations evolved metabolic resistance to trifluralin, and at least four of them also possessed the known and/or putative target-site mutations. These results confirm that a high incidence of resistance to the dinitroaniline herbicide (trifluralin) is present, and target-site tubulin mutations make a major contribution to resistance in these annual ryegrass populations. Co-evolution of both target-site and non-target-site resistance to per-emergence herbicides warrants diverse management tactics.

Abstract P246: Discovery and characterization of selective, FGFR1 sparing, inhibitors of FGFR2/3 oncogenic mutations for the treatment of cancers

Abstract Background: Targeting FGFR genetic alterations using small molecule inhibitors is a validated therapeutic strategy for urothelial carcinoma and cholangiocarcinoma. However, the current FDA-approved pan-FGFR inhibitors, erdafitinib and pemigatinib, are subject to FGFR1-mediated dose-limiting toxicities (e.g., hyperphosphatemia). These treatments necessitate a high rate of dose reductions, interruptions, and discontinuations, thereby potentially limiting efficacy. In addition, drug-resistant mutations (e.g., gatekeeper) in FGFR2 and FGFR3 genes rapidly emerge in patients treated with these drugs. Our research goals are to reveal the full spectrum of oncogenic FGFR2 and FGFR3 mutations that drive tumor growth and to discover an inhibitor that selectively targets these mutations together with FGFR2 and FGFR3 gene fusion and drug-resistance mutations, while minimizing FGFR1 activity and associated toxicities. We hypothesize that this will deliver an FGFR precision medicine with enhanced anti-tumor activity, an improved drug resistance profile, and broader mutational coverage. Methods: Applying the Mutation-Allostery-Pharmacology (MAP) platform technology developed by Black Diamond Therapeutics, we defined a spectrum of 34 allosteric FGFR2/3 oncogenic mutations, including over 28 previously uncharacterized mutations that we now show to be oncogenic. The MAP platform allowed us to further classify those mutations into functional clusters or families of mutations that can be targeted using a single compound. While located throughout the extracellular and kinase domains, we demonstrated how these functional clusters activate FGFR2 or FGFR3. Among the mechanisms identified, a functional cluster of mutations is activated due to disulfide-bond mediated dimerization. Results: Herein, we report the discovery of a series of orally available, selective FGFR2/3 inhibitors that 1) shows antiproliferative potency across all 34 mutations; 2) spares FGFR1-wild-type; 3) is active against gatekeeper mutations and 4) shows favorable selectivity versus a subset of closely related kinases in the human kinome. In addition to being potent against FGFR2 and FGFR3 primary mutations, we demonstrated that our FGFR1 sparing inhibitors retain potency against the most prevalent FGFR2 resistant mutations. When dosed orally, one example was well tolerated and exhibited dose-dependent PK/PD and anti-tumor efficacy and regression in several FGFR2 and FGFR3 driven xenograft models in mice. In addition, when dosed at efficacious doses, no FGFR1-mediated hyperphosphatemia was observed in these animals instead of the animals that were treated with pan FGFR inhibitors. Conclusion: Our data support the development of rationally designed selective inhibitors targeting a spectrum of FGFR2/3 mutations while sparing dose limiting FGFR1 activity. Citation Format: Etienne Dardenne, Fernando Padilla, Sara Rasmussen, Shao Ning Yang, Ahmet Mentes, Luisa Shin Ogawa, Anthony Trombino, Darlene Romashko, Maria Chevtsova, Shalabh Thakur, Elisabeth Buck, Christopher Roberts, Matthew Lucas, Tai-An Lin. Discovery and characterization of selective, FGFR1 sparing, inhibitors of FGFR2/3 oncogenic mutations for the treatment of cancers [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P246.

Novel potential oncogenic and druggable mutations of FGFRs recur in the kinase domain across cancer types

Fibroblast growth factor receptors (FGFRs) are recurrently altered by single nucleotide variants (SNVs) in many human cancers. The prevalence of SNVs in FGFRs depends on the cancer type. In some tumors, such as the urothelial carcinoma, mutations of FGFRs occur at very high frequency (up to 60%). Many characterized mutations occur in the extracellular or transmembrane domains, while fewer known mutations are found in the kinase domain. In this study, we performed a bioinformatics analysis to identify novel putative cancer driver or therapeutically actionable mutations of the kinase domain of FGFRs. To pinpoint those mutations that may be clinically relevant, we exploited the recurrence of alterations on analogous amino acid residues within the kinase domain (PK_Tyr_Ser-Thr) of different kinases as a predictor of functional impact. By exploiting MutationAligner and LowMACA bioinformatics resources, we highlighted novel uncharacterized mutations of FGFRs which recur in other protein kinases. By revealing unanticipated correspondence with known variants, we were able to infer their functional effects, as alterations clustering on similar residues in analogous proteins have a high probability to elicit similar effects. As FGFRs represent an important class of oncogenes and drug targets, our study opens the way for further studies to validate their driver and/or actionable nature and, in the long term, for a more efficacious application of precision oncology.

Functional consequences of TCF4 missense substitutions associated with Pitt-Hopkins syndrome, mild intellectual disability, and schizophrenia

Transcription factor 4 (TCF4) is a basic helix-loop-helix transcription factor essential for neurocognitive development. The aberrations in TCF4 are associated with neurodevelopmental disorders including schizophrenia, intellectual disability, and Pitt-Hopkins syndrome, an autism-spectrum disorder characterized by developmental delay. Several disease-associated missense mutations in TCF4 have been shown to interfere with TCF4 function, but for many mutations, the impact remains undefined. Here, we tested the effects of 12 functionally uncharacterized disease-associated missense mutations and variations in TCF4 using transient expression in mammalian cells, confocal imaging, in vitro DNA-binding assays, and reporter assays. We show that Pitt-Hopkins syndrome-associated missense mutations within the basic helix-loop-helix domain of TCF4 and a Rett-like syndrome-associated mutation in a transcription activation domain result in altered DNA-binding and transcriptional activity of the protein. Some of the missense variations found in schizophrenia patients slightly increase TCF4 transcriptional activity, whereas no effects were detected for missense mutations linked to mild intellectual disability. We in addition find that the outcomes of several disease-related mutations are affected by cell type, TCF4 isoform, and dimerization partner, suggesting that the effects of TCF4 mutations are context-dependent. Together with previous work, this study provides a basis for the interpretation of the functional consequences of TCF4 missense variants.

Rare and novel GNAS gene mutations in Chinese patients with thyroid cancer

AbstractBackgroundA large number of novel, rare, and uncharacterized mutations in Tumors including thyroid cancer (TC) have been discovered. In this study, we reported two GNAS mutations in Chinese TC patients, and assessed the functional impact.MethodsTwo hundred and twenty five formalin‐fixed, paraffin‐embedded (FFPE) tissue specimens from Chinese TC patients were tested by NGS. The functional impact of GNAS mutations was assessed using sorting intolerant from tolerant tools (SIFT), polymorphism phenotyping v2 (POLYPHEN‐2) and the mutation assessor. The GNAS protein structure was modeled by Iterative Threading ASSEmbly Refinement (I‐TASSER), and modeling of mutations was performed and visualised by PyMOL.ResultsTwo somatic missense GNAS gene mutations including a rare c.956A>G(p.D319G) and a novel c.1399G>T(p.A467S) were detected in 225 TC tissues (2/225, 0.89%). The novel GNAS mutation c.1399G>T(p.A467S) was predicted to benign and low impact on the protein. SIFT and Mutation Assessor predicted c.956A>G(p.D319G) will be neutral and have low impact, while Polyphen‐2 indicated the mutation may be probably damaging. c.1399G>T(p.A467S) and c.956A>G(p.D319G) molecular modeling data showed two mutations not changed in Gsα, and showed little predicted effect on protein structure.ConclusionsA rare somatic GNAS gene mutations c.956A>G(p.D319G) and a novel somatic GNAS mutation c.1399G>T(p.A467S) were found in the present study, which appeared to be associated with low impact on the protein.

35MO Discovery and characterization of selective, FGFR1-sparing, inhibitors of FGFR2/3 oncogenic mutations for the treatment of cancers

Targeting FGFR genetic alterations using small molecule inhibitors is a validated therapeutic strategy for urothelial carcinoma and cholangiocarcinoma. However, the current FDA-approved pan-FGFR inhibitors, erdafitinib and pemigatinib, are subject to FGFR1-mediated dose-limiting toxicities (e.g., hyperphosphatemia). These treatments necessitate a high rate of dose reductions, interruptions, and discontinuations, thereby potentially limiting efficacy. In addition, drug-resistant mutations (e.g., gatekeeper) in FGFR2 and FGFR3 genes rapidly emerge in patients treated with these drugs. Our research goals are to reveal the full spectrum of oncogenic FGFR2 and FGFR3 mutations that drive tumor growth and to discover an inhibitor that selectively targets these mutations together with FGFR2 and FGFR3 gene fusion and drug-resistance mutations, while minimizing FGFR1 activity and associated toxicities. We hypothesize that this will deliver an FGFR precision medicine with enhanced anti-tumor activity, an improved drug resistance profile, and broader mutational coverage. Applying the Mutation-Allostery-Pharmacology (MAP) platform technology developed by Black Diamond Therapeutics, we define a spectrum of 20 allosteric FGFR2/3 oncogenic mutations, including 10 previously uncharacterized mutations that we now show to be oncogenic. We demonstrated how this mutation family is activated due to disulfide-bond mediated dimerization; hence we term these mutations locked-dimer (Lo-Di) FGFR oncogenes. Herein, we report the discovery of a series of orally available, selective Lo-Di-FGFR2/3 inhibitors that 1) shows antiproliferative potency across all 20 mutations; 2) spares FGFR1-wild-type; 3) is active against gatekeeper mutations and 4) shows favorable selectivity versus a subset of closely related kinases in the human kinome. When dosed orally, one example was well tolerated and exhibited dose-dependent PK/PD and anti-tumor efficacy and regression in the UM-UC-14 xenograft model in mice. Our data support the development of rationally designed selective inhibitors targeting a spectrum of FGFR2/3 mutations while sparing dose limiting FGFR1 activity.

Functional characterization of SMARCA4 variants identified by targeted exome-sequencing of 131,668 cancer patients

Genomic studies performed in cancer patients and tumor-derived cell lines have identified a high frequency of alterations in components of the mammalian switch/sucrose non-fermentable (mSWI/SNF or BAF) chromatin remodeling complex, including its core catalytic subunit, SMARCA4. Cells exhibiting loss of SMARCA4 rely on its paralog, SMARCA2, making SMARCA2 an attractive therapeutic target. Here we report the genomic profiling of solid tumors from 131,668 cancer patients, identifying 9434 patients with one or more SMARCA4 gene alterations. Homozygous SMARCA4 mutations were highly prevalent in certain tumor types, notably non-small cell lung cancer (NSCLC), and associated with reduced survival. The large sample size revealed previously uncharacterized hotspot missense mutations within the SMARCA4 helicase domain. Functional characterization of these mutations demonstrated markedly reduced remodeling activity. Surprisingly, a few SMARCA4 missense variants partially or fully rescued paralog dependency, underscoring that careful selection criteria must be employed to identify patients with inactivating, homozygous SMARCA4 missense mutations who may benefit from SMARCA2-targeted therapy.

Effect of Mutated ids Overexpression on IDS Enzyme Activity and Developmental Phenotypes in Zebrafish Embryos: A Valuable Index for Assessing Critical Point-Mutations Associated with Mucopolysaccharidosis Type II Occurrence in Humans.

Mucopolysaccharidosis type II (MPS II) is an X-linked disorder resulting from a deficiency in iduronate 2-sulfatase (IDS), which is reported to be caused by gene mutations in the iduronate 2-sulfatase (IDS) gene. Many IDS mutation sites have not yet had their causal relationship with MPS II characterized. We employed a gain-of-function strategy whereby we microinjected different mutated zebrafish ids (z-ids) mRNAs corresponded to human IDS gene into zebrafish embryos, and then measured their total IDS enzymatic activity and observed the occurrence of defective phenotypes during embryonic development. We examined three known mutation sites for human IDS genes (h-IDS) associated with MPS II symptoms, including h-IDS-P86L, -S333L and -R468W, which corresponded to z-ids-P80L, -S327L and -R454W. When these three mutated z-ids mRNAs were overexpressed in zebrafish embryos, the IDS enzymatic activity of the total proteins extracted from the injected embryos was not increased compared with the endogenous IDS of the untreated embryos, which suggests that the IDS enzymatic activity of these three mutated z-ids was totally lost, as expected. Additionally, we observed defective phenotypes in these injected embryos, resulting from the failed IDS enzyme breakdown, which, in turn, has a dominant negative effect on the endogenous wild-type IDS function. These phenotypes were similar to the clinical symptoms observed in MPS II pathogenesis. We further studied six uncharacterized IDS mutation sites as identified by the Taiwanese MPS newborn screening programs. We propose a novel IDS enzyme activity assay combined with phenotypic observation in zebrafish embryos, as an alternative platform for quickly providing a valuable index for preliminarily assessment of any identified IDS point mutation gene that has not yet been characterized, in the context of its role in MPS II development.

A novel variant of VEGFR2 identified by a pan-cancer screening of recurrent somatic mutations in the catalytic domain of tyrosine kinase receptors enhances tumor growth and metastasis.

In cancer genomics, recurrence of mutations in gene families that share homologous domains has recently emerged as a reliable indicator of functional impact and can be exploited to reveal the pro-oncogenic effect of previously uncharacterized variants. Pan-cancer analyses of mutation hotspots in the catalytic domain of a subset of tyrosine kinase receptors revealed that two infrequent mutations of VEGFR2 (R1051Q and D1052N) recur in analogous proteins and correlate with reduced patient survival. Functional validation showed that both R1051Q and D1052N mutations increase the enzymatic activity of VEGFR2. The expression of VEGFR2R1051Q potentiates the PI3K/Akt signaling axis in cancer cells, increasing their tumorigenic potential in vitro and in vivo. In addition, it confers to cancer cells an increased sensitivity to the VEGFR2-targeted tyrosine kinase inhibitor Linifanib. In the context of an efficacious application of anti-cancer targeted therapies, these findings indicate that the screening for uncharacterized mutations, like VEGFR2R1051Q, may help to predict patient prognosis and drug response, with significant clinical implications.